Optimized buffers and conditions to achieve high coverage of GC-rich regions

The QIAseq targeted DNA Panels have been developed as a complete Sample to Insight solution to enable digital DNA sequencing by utilizing molecular barcodes. Digital DNA sequencing is a unique approach to detect low-frequency variants with high confidence by overcoming the issues of PCR duplicates, false positives and library bias.

Each panel is a one-box, NGS platform-agnostic solution that contains all the necessary components to construct libraries from enriched genomic targets. Primer design is based on single primer extension, in which each genomic target is enriched by one target-specific primer and one universal primer – a strategy that removes conventional two target-specific primer design restriction and reduces the amount of required primers. All primers required for a panel are pooled into an individual primer pool to reduce panel handling and the number of pools required for enrichment and library construction. Platform-specific indexes, which are contained in a separate box, allow the multiplexing of up to 384 samples per sequencing run.

QIAseq Targeted DNA Panels are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease.

Workflow

Isolated DNA, as low as 20 ng, is enzymatically fragmented to generate small pieces of dsDNA. This is followed by the library construction step, in which IL-N7 adapters, molecular barcodes, and sample indexes are incorporated into DNA fragments generated in the previous step. Library fragments now serve as templates for target enrichment using single primer extension. In this step, targets are enriched using a single gene-specific primer and a universal forward primer. The final step is library amplification and sample indexing (for dual indexing) using the IL-S5 sample index primer and a universal primer.

Coverage of GC-rich genomic regions

The QIAseq DNA panels use a proprietary buffer mixture to efficiently sequence GC-rich regions within the genome. Two examples are shown here: CEBPA and CCND1. Complete coverage of exonic regions within those two genes is achieved.

Principle of molecular barcodes

A variant identified in a sample represents one of two events: a true or false variant. False variants can be introduced at any step during the workflow, including sequencing reactions. This results in the inability to accurately and confidently call rare variants (those present at 1% of the sample). Due to PCR duplicates generated in amplification steps, all DNA fragments look exactly the same, and there is no way to tell whether a specific DNA fragment is a unique DNA molecule or a duplicate of a DNA molecule. With molecular barcodes, since each unique DNA molecule is barcoded before any amplification takes place, unique DNA molecules are identified by their unique barcodes, and PCR duplicates carrying the same barcode are removed, thereby increasing the sensitivity of the panel.

Uniformity

The QIAseq targeted DNA panels deliver outstanding sequencing metrics. 6000 SNPs were enriched from 20 ng of NA12878 DNA. Library was constructed for sequencing on a MiSeq, with 4,000,000 reads generated. The panel achieved a uniformity of 99.5% at 0.2x of mean coverage, and 96% at 0.5x of mean coverage.

Sensitivity: Digital DNA sequencing approach is optimized to deliver high confidence in calling low-frequency DNA variants. Over 90% sensitivity for 1% NA12878 SNP and indel on typical coding region with false positive less than 15 per mega base region when variants are detected with tiled primer design to cover complete coding region of each gene.

Universality: The chemistry used in the QIAseq targeted DNA panels and workflow is compatible with both regular and GC-rich genomic regions, allowing one to achieve 100% coverage of genes rich in GC content such as CEBPA and CCND1 (see figure: coverage of GC-rich genomic regions)

Flexibility: The QIAseq targeted DNA panels offer a high degree of flexibility in content and sample multiplexing. Several cataloged panels have been developed for a wide range of applications. One can also build a custom panel for a specific content, or extend the contents of an existing cataloged panel. Up to 384 samples can be multiplexed using the QIAseq indexes.

Principle

PCR duplicates are a major issue in targeted DNA sequencing, since, through PCR amplification, they turn unique DNA molecules into identical DNA molecules that cannot be distinguished from each other. In addition, errors from PCR amplification and sequencing process may also be present in final reads that lead to false positive variants in sequencing results. This, in turn, results in the inability to confidently call DNA variants present at low frequencies in the starting DNA material. To overcome the issue of PCR duplicates and amplification artifacts, the QIAseq Targeted DNA Panels use digital sequencing by incorporating molecular barcodes into the starting DNA material before any amplification takes place, thereby preserving the uniqueness of the starting DNA molecules and overcoming the issues of PCR duplicates, false positives and library bias.

Procedure

The entire workflow of the QIAseq targeted DNA panels to go from extracted DNA to sequencing-ready libraries can be completed in 9 hours (see figure Workflow). Extracted DNA is fragmented, genomic targets are molecularly barcoded and enriched, and libraries are constructed. Sequencing files can be fed into the QIAseq pipeline, a cloud-based data analysis pipeline, which will filter, map and align reads, as well as count unique molecular barcodes associated with targeted genomic regions, and call variants with a barcode-aware algorithm. This data can then be fed into IVA or QCI for interpretation.

Applications

The QIAseq targeted DNA panels can be used to call a variety of DNA variants from a wide range of sample types for numerous applications.